Rice having short anther characteristics, and method for producing the same

Abstract

To provide a method for producing a rice having an anther-shortening trait.SOLUTION: A method for producing a rice having an anther-shortening trait includes the step of artificially inhibiting the functions of at least one gene selected from the group consisting of genes encoding a protein having a specific amino acid sequence in rice.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to rice having a short anther trait and a method for producing the same.

Background Art

[0002] Although rice is basically a plant that self-pollinates frequently, it is known that it also cross-pollinates at a low frequency because pollen scatters outside after flowering.

[0003] Therefore, among producers and consumers, there is a strong concern about the cross-breeding and mixing of genetically modified crops with surrounding general cultivated crops, which is a major obstacle in the practical application of genetically modified crops. In order to prevent cross-breeding with surrounding wild plants and the same species of crops due to pollen scattering, measures such as ensuring a cultivation distance, adjusting the cultivation period so that the flowering periods do not overlap, flower picking, emasculation, bagging of flowers, and installation of windbreak nets are carried out, but all of these require a great deal of labor.

[0004] Also, in the rice seed production field, the mixing of different varieties is a major problem, and a lot of labor is required for the discovery and removal of different strains generated by natural cross-breeding. This is a great burden on rice seed production farmers who are facing an aging population and a shortage of labor.

[0005] From the above, a method for suppressing cross-breeding without much labor is required. As such a method, for example, the use of rice having a trait of pollinating without flowering (closed-flower pollinating rice) is considered. If rice varieties such as rice having closed-flower pollination can be put into practical use, it is expected that the maintenance of variety purity can be achieved at a lower cost and that it can contribute to the sustainable and stable maintenance of seed production.

[0006] Under such a background, attempts have been made to put into practical use a mutant spw1-cls1 having closed-flower pollination due to the incomplete formation of lodicules, which are organs involved in flowering (Patent Document 1). However, it has been clarified that the mutant flowers during the floret formation period at low temperatures, and the development of new cross-breeding suppression technologies is desired.

[0007] In this regard, san-1 (short anther) is a mutant induced by a chemical mutagenic substance using T65 as the original variety, and exhibits short anthers, with an anther length reduced by approximately 30% compared to the wild type (Non-Patent Literature 1). Due to the shortness of the anthers, they are difficult to extract from the glumes, and pollen is also less likely to be dispersed, so it is expected to be used as a new cross-pollination suppression technology.

[0008] However, the causative gene involved in the phenotype of this mutant has not yet been identified. Therefore, it has been difficult to create lines with this short-anther trait in varieties other than T65, and consequently, to use such lines to suppress crossbreeding. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2007-300876 [Non-patent literature]

[0010] [Non-Patent Document 1] Research Results Report for the 2016 Grant-in-Aid for Scientific Research, Principal Investigator: Hitoshi Yoshida, Research Project Title: Elucidation of the Control Mechanism for Rice Flower Organ Size, Publication Date: March 22, 2018 [Overview of the project] [Problems that the invention aims to solve]

[0011] This invention has been made in view of the above-mentioned problems, and aims to identify the causative gene involved in the short anther trait in san-1 and to provide a method for producing rice having the short anther trait by targeting said gene. [Means for solving the problem]

[0012] To achieve the above objective, the inventors first crossed a rice mutant san-1 possessing the short anther trait with the rice variety Casalas and performed map-based cloning using the F2 population. As a result, the causative gene was predicted to be located between markers RM18639 and RM6841 on chromosome 5. Further narrowing down the candidate region, it was predicted that the candidate region was located between markers IRIC11 and RM18719. Twenty genes were found within this candidate region in the RAP-DB database. Next, by comparing the gene sequences, it was found that in san-1, the 236th guanine from the translation start site of the gene Os05g0421300 was replaced with adenine, resulting in the formation of an immature stop codon.

[0013] Therefore, guide RNAs were designed at two different locations in the first exon of the candidate gene (the gene encoding the amino acid sequence described in SEQ ID NO: 2), and frameshift mutations were induced using genome editing with the CRISPR / Cas9 method. Furthermore, frameshift mutations were also induced in the second exon using genome editing. As a result, since the short anther characteristic similar to that of san-1 was observed in both guide RNAs, it was finally revealed that this gene is the causative gene involved in the short anther trait in san-1, thus completing the present invention. Accordingly, the present invention provides the following. <1> A method for producing rice having the short anther trait, A method comprising the step of artificially suppressing the function of at least one gene in rice selected from the group consisting of (a) to (d) below. (a) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 2. (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence described in Sequence ID No. 2. (c) A gene encoding an amino acid sequence having 95% or more homology to the amino acid sequence described in Sequence ID No. 2. (d) A gene containing DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence encoding the amino acid sequence described in Sequence ID No. 2. <2> Rice with a short anther trait, in which the function of at least one gene selected from the group consisting of (a) to (d) below is artificially suppressed. (a) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 2. (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence described in Sequence ID No. 2. (c) A gene encoding an amino acid sequence having 95% or more homology to the amino acid sequence described in Sequence ID No. 2. (d) A gene containing DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence encoding the amino acid sequence described in Sequence ID No. 2. [Effects of the Invention]

[0014] According to the present invention, it is possible to produce rice with short anthers. Because of its short anthers, the anthers of this rice are difficult to extract from the glumes, and the pollen is also less likely to be dispersed. Therefore, it is possible to suppress cross-pollination with other rice varieties. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows that the rice mutant san-1 possesses the short anther trait. In the figure, (A) is a photograph showing the results of observing the glumes with the outer glume removed for the wild type (T65) and san-1. (B) is a graph showing that the anther length of san-1 is shortened by about 30% compared to the wild type. (C) is a graph showing that there is no significant difference in glume length between the wild type and san-1. [Figure 2]This is a schematic diagram showing the process of mapping the causal gene in san-1. Between markers IRIC11 and RM18719, which are candidate regions for the causal gene, the gene regions predicted in the RAP-DB database are indicated by arrows. Among these 20 genes, gene Os05g0421300 was identified as the causal gene for san-1. [Figure 3] This is a diagram showing an overview of the base substitution found in the causal gene of san-1. The 236th guanine (G) counted from the translation start point of gene Os05g0421300 was substituted with adenine (A), causing the codon that originally encoded tryptophan (W) to change to a premature stop codon. [Figure 4] This is a schematic diagram showing the structure of the rice SAN gene. The rice SAN gene encodes 601 amino acids with an ARM (Armadillo repeat) domain on the N-terminal side and a TPR (tetratricopeptide repeat) domain on the C-terminal side. [Figure 5] This is a diagram showing an overview of the rice genome editing vector. [Figure 6] This is a schematic diagram showing the base insertion site in the rice san-CR1 genome editant. A thymine insertion occurred between the 392nd and 393rd bases counted from the translation start point of the rice SAN gene, resulting in a frameshift. [Figure 7] This is a schematic diagram showing the base insertion site in the rice san-CR2 genome editant. A thymine insertion occurred between the 153rd and 154th bases counted from the translation start point of the rice SAN gene, resulting in a frameshift. [Figure 8] This is a photograph showing the results of observing short anthers in the rice SAN genome editants. Short anther phenotypes similar to those of san-1 were observed in both the san-CR1 and san-CR2 genome editants. [Figure 9] This is a schematic diagram showing the base insertion site in the rice san-CR3 genome editant. A thymine insertion occurred between the 1667th and 1668th bases counted from the translation start point of the rice SAN gene, resulting in a frameshift. [Figure 10] These are photographs and graphs showing the results of observing anther shortening in rice SAN-CR3 genome edited plants. The san-CR3#3 genome edited plant (#5) showed weaker anther shortening than the san-CR2 edited plant (#14-1). [Modes for carrying out the invention]

[0016] (Method for producing rice with short anther characteristics) As shown in the examples described below, the inventors selected a candidate gene (a gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2) involved in the phenotype of san-1 (short anther), a rice line possessing the short anther trait, by map-based cloning or the like. Furthermore, they succeeded in conferring the short anther trait to wild-type rice by suppressing the function of the said gene using genome editing. Therefore, the method for producing rice possessing the short anther trait of the present invention is characterized by including a step of artificially suppressing the function of the said gene (short anther gene), and more specifically provides the following.

[0017] A method for producing rice having the short anther trait, A method comprising the step of artificially suppressing the function of at least one gene in rice selected from the group consisting of (a) to (d) below. (a) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 2. (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence described in Sequence ID No. 2. (c) A gene encoding an amino acid sequence having 95% or more homology to the amino acid sequence described in Sequence ID No. 2. (d) A gene containing DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence encoding the amino acid sequence described in Sequence ID No. 2.

[0018] In the present invention, the "short anther trait" refers to a trait in which the length of the anther of the stamen is shortened. Here, "anther length" refers to the length of the longitudinal axis of the anther, which is approximately elliptical in shape. Furthermore, "shortening" means, for example, that the length becomes 10% or more (preferably 20% or more, more preferably 30% or more) compared to before artificially suppressing the function of the short anther gene according to the present invention (for example, wild-type rice).

[0019] In the present invention, there are no particular restrictions on the "rice" to which the short anther trait is to be conferred, as long as it is a plant belonging to the genus Oryza in the family Poaceae. For example, it may be a Japonica variety or an Indica variety. It may also be a wild variety or a cultivated variety. Furthermore, these rice varieties may be genetically modified or genome-edited (for example, disease-resistant crops, herbicide-resistant crops, insect-resistant crops, taste-enhanced crops, storability-enhanced crops, yield-enhanced crops). Thus, in the present invention, there are no particular restrictions on the rice to which the short anther trait is to be conferred, but from the viewpoint of further suppressing cross-pollination, rice with cleistogamous pollination is preferred. More specifically, examples of such rice include spw1-cls1 and spw1-cls2 (Lombardo F. et al., Plant Biotechnol. J., 2017, Vol. 15, pp. 97-106).

[0020] Examples of genes encoding typical amino acid sequences that are targeted for functional suppression in this invention are shown in Table 1 below.

[0021] [Table 1]

[0022] It should be noted that mutations in nucleotide sequences can occur in nature. Consequently, the encoded amino acids can also change. Therefore, the short-anther genes of the present invention also include genes encoding proteins consisting of amino acid sequences in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence described in Sequence ID No. 2, as long as their function is suppressed to confer the short-anther trait.

[0023] Here, "multiple" usually refers to 120 amino acids or less, preferably 90 amino acids or less, more preferably 60 amino acids or less, even more preferably 55 amino acids or less, even more preferably 50 amino acids or less, even more preferably 45 amino acids or less, even more preferably 40 amino acids or less, even more preferably 35 amino acids or less, even more preferably 30 amino acids or less (for example, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less), and particularly preferably 10 amino acids or less (for example, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids).

[0024] Furthermore, with the current level of technology, a person skilled in the art can, once a specific gene is obtained, use its nucleotide sequence information to identify its homologous gene from the same plant species. Methods for identifying homologous genes include, for example, hybridization techniques (Southern, EM, J. Mol. Biol., 98:503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, RK, et al. Science, 230:1350-1354, 1985; Saiki, RK et al. Science, 239:487-491, 1988). To identify homologous genes, hybridization reactions are usually performed under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5x SSC, or hybridization conditions with equivalent stringency. Using conditions with higher stringency, such as 6M urea, 0.4% SDS, and 0.1x SSC, it is possible to isolate genes with higher homology. The short anther genes of the present invention include genes that, insofar as their function is suppressed, can confer the short anther trait, and which include DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 2 (for example, the nucleotide sequence described in SEQ ID NO: 1).

[0025] The proteins encoded by the identified homologous genes typically have high homology (high similarity), preferably high identity, with those encoded by the specific genes. Here, "high" means at least 80%, preferably 85%, more preferably 90%, and even more preferably 95% (for example, 96%, 97%, 98%, 99%). The short anther genes of the present invention include genes that encode amino acid sequences having 95% or more homology (similarity) or 95% or more identity with, for example, the amino acid sequence described in Sequence ID No. 2, insofar as their function can confer the short anther trait.

[0026] Sequence homology can be determined using the BLAST program (Altschul et al. J.Mol.Biol., 215:403-410, 1990). This program is based on the BLAST algorithm by Karlin and Altschul (Proc.Natl.Acad.Sci.USA, 87:2264-2268, 1990, Proc.Natl.Acad.Sci.USA, 90:5873-5877, 1993). For example, when analyzing amino acid sequences using BLAST, the parameters should be, for example, score=50 and wordlength=3. When analyzing amino acid sequences using the Gapped BLAST program, it can be done as described by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using the BLAST and Gapped BLAST programs, use the default parameters of each program. The specific methods for these analyses are publicly known.

[0027] The "artificial suppression of short anther gene function" of the present invention includes both complete suppression (inhibition) and partial suppression of said function. In addition to artificial suppression of short anther gene expression, it also includes artificial suppression of the activity of the protein encoded by the short anther gene. Such artificial suppression can be achieved, for example, by introducing mutations into the coding region, non-coding region, or transcriptional regulatory region (promoter region, etc.) of the short anther gene.

[0028] In the present invention, there are no particular restrictions on the mutations introduced into the short anther gene, as long as they suppress the function of the gene. Examples include nucleotide substitutions, deletions, additions, and / or insertions, but frameshift mutations, nonsense mutations, null mutations, in-frame mutations, inversions, and translocations are preferred. Furthermore, in the present invention, the mutations introduced into the short anther gene may also be mutations in epigenetic regulation that do not involve such nucleotide mutations. Examples of epigenetic regulation include DNA methylation and histone chemical modifications (acetylation, methylation, phosphorylation, ubiquitination, etc.). There are also no particular restrictions on the number of mutations introduced into the short anther gene, as long as they suppress the function of the gene. It may be one mutation or multiple mutations (for example, two, three or fewer, five or fewer, ten or fewer, twenty or fewer, thirty or fewer, forty or fewer, fifty or fewer).

[0029] Examples of such mutations include, as shown in the examples below, nucleotide mutations involving changes or deletions of amino acids from position 557 onwards (approximately 7% of the total) in the amino acid sequence described in SEQ ID NO: 2, nucleotide mutations involving changes or deletions of amino acids from position 131 onwards (approximately 78% of the total) in the amino acid sequence described in SEQ ID NO: 2, nucleotide mutations involving changes or deletions of amino acids from position 79 onwards (approximately 87% of the total) in the amino acid sequence described in SEQ ID NO: 2, and nucleotide mutations involving changes or deletions of amino acids from position 52 onwards (approximately 91% of the total) in the amino acid sequence described in SEQ ID NO: 2. Such deletions suppress the function of the short anther gene of the present invention, making it possible to obtain rice with the short anther trait.

[0030] Therefore, the mutation introduced into a short anther gene does not need to result in the loss of the entire amino acid sequence of the protein encoded by that gene; it may be introduced into the gene in such a way that only a portion of the sequence is lost or altered.

[0031] In addition, when a portion of the expressed protein is deleted due to a gene mutation, it is usually sufficient for 5% or more of the total amino acids (for example, 6% or more, 7% or more, 8% or more, or 9% or more) to be changed or deleted, preferably 10% or more, more preferably 20% or more, even more preferably 25% or more, more preferably 30% or more, even more preferably 35% or more, more preferably 40% or more, even more preferably 45% or more, more preferably 50% or more, even more preferably 55% or more, more preferably 60% or more, even more preferably 65% ​​or more, more preferably 70% or more, even more preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and even more preferably 95% or more (for example, 96% or more, 97% or more, 98% or more, or 99% or more) to be changed or deleted.

[0032] There are no particular limitations on the regions in which the amino acid sequence is altered or deleted, but for example, as shown in the examples described later, the C-terminal region can be cited. In addition, regions in the amino acid sequence encoded by the short anserine gene of the present invention that are suggested to be important for exerting their function (protein-protein interactions, etc.) (ARM (armadillo repeat) domain and / or TPR (tetratricopeptide repeat)) can also be cited as examples of regions in which the amino acid sequence is altered or deleted.

[0033] Furthermore, in the present invention, it is possible to control the degree of anther shortening by adjusting the site of mutation introduction in the short anther gene, the type of mutation introduced, or the region in the amino acid sequence that is changed or deleted as a result. For example, as shown in the examples described later, strong anther shortening is brought about by introducing a mutation upstream of the short anther gene (the region corresponding to positions 1 to 400 in the coding amino acid sequence), while weak anther shortening is brought about by introducing a mutation downstream (the region corresponding to positions 401 to 601 in the coding amino acid sequence). It is also possible to bring about weak anther shortening by weakening the activity of the coding protein through in-frame mutations, etc.

[0034] The introduction of mutations into short anserine genes can be achieved by mutation introduction methods known to those skilled in the art. Such known methods include, but are not limited to, genome editing, physical mutation introduction, methods using chemical mutagens, methods introducing transposons into genomic DNA, and methods targeting transcripts using siRNA, antisense RNA, and RNA with ribozyme activity.

[0035] Among these methods, genome editing, methods targeting transcripts, and the tilling method described later are preferred from the perspective of being able to artificially introduce mutations by targeting short anther genes.

[0036] Genome editing is a method of modifying target genes using site-specific nucleases (e.g., zinc finger nucleases (ZFNs), transcription-activating effector nucleases (TALENs), and DNA double-strand cleavage enzymes such as CRISPR-Cas enzymes). For example, fusion proteins such as ZFNs (US Patents 6,265196, 8,524500, 7,888121, European Patent 1,720995), TALENs (US Patents 8,470973, 8,586363), PPR (pentatricopeptide repeat) with a fused nuclease domain (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (US Patent 8,697359, International Publication 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)), and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and Examples include methods using guide RNA-protein complexes, such as those described in "vertebrate adaptive immune systems" (Science, DOI:10.1126 / science.aaf8729, (2016)), or protein complexes.

[0037] There are no particular restrictions on the "Cas enzyme," and it can be appropriately selected depending on the purpose. Examples include type I CRISPR enzymes, type II CRISPR enzymes, type III CRISPR enzymes, etc., but type II CRISPR enzyme Cas9 is preferred. There are no particular restrictions on the "Cas9," and it can be appropriately selected depending on the purpose. Examples include Cas9 from Streptococcus pneumoniae, Cas9 from Streptococcus pyogenes, Cas9 from S. thermophilus, Cas9 from Staphylococcus aureus, etc., but Cas9 from Streptococcus pyogenes (SpCas9) is preferred. Furthermore, the Cas9 mutant derived from these organisms may also be a Cas9 D10A mutant known to function as a nickase (a DNA-cutting enzyme that inserts a nick into only one DNA strand), or it may be a Cas9 homolog or orthologue.

[0038] Examples of physical mutagenesis methods include heavy ion beam (HIB) irradiation, fast neutron irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0039] Methods using chemical mutagens include, for example, treating seeds with chemical mutagens (see Zwar and Chandler, Planta, 1995, Vol. 197, pp. 39-48, etc.). There are no particular restrictions on the chemical mutagens, but examples include N-methyl-N-nitrosourea (MNU), ethylmethanesulfate (EMS), N-ethyl-N-nitrosourea (ENU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrite, formic acid, and nucleotide analogs.

[0040] Methods for introducing transposons, etc., into genomic DNA include, for example, T OS Methods include inserting transposons such as 17, T-DNA, etc., into the plant's genomic DNA (see Kumar et al., Trends Plant Sci., 2001, Vol. 6, No. 3, pp. 127-134, and Tamara et al., Trends in Plant Science, 1999, Vol. 4, No. 3, pp. 90-96).

[0041] For rice plants in which mutations have been introduced using the methods described above, the presence of mutations in the short anther gene can be confirmed by known methods. Examples of such known methods include DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, Southern blotting, and Northern blotting. Using these methods, it is possible to determine whether or not a mutation has been introduced in the short anther gene by comparing the sequence or length of the gene before and after the introduction of the mutation. Furthermore, by using Northern blotting, RT-PCR, Western blotting, ELISA, microarray analysis, etc., if a decrease in the expression level of the transcript or translation product of the short anther gene is observed in rice plants in which mutations have been introduced in the transcriptional regulatory region, it can be confirmed that the rice plants have had mutations introduced in the short anther gene.

[0042] Another method for confirming the introduction of mutations into short anther genes is TILLING (Targeting Induced Local Lesions in Genomes) (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when non-selective mutations are introduced into the rice genome using heavy ion beam irradiation or chemical mutagens, the short anther gene or a part thereof can be amplified by PCR, and then individuals with mutations in the amplified product can be selected by TILLING or the like.

[0043] Furthermore, by crossbreeding rice plants into which mutations have been introduced using the method described above with wild-type rice plants and then performing a backcross, it is possible to remove mutations introduced into genes other than the target gene.

[0044] Rice plants in which the function of a short anther gene is suppressed by introducing a mutation into the short anther gene may be heterozygotes of the short anther gene. In such cases, for example, homozygotes possessing the short anther gene into which the mutation was introduced can be selected from the F1 plants by crossing such heterozygotes together to obtain F1 plants. In this case, "rice plants that are homozygotes possessing the short anther gene into which the mutation was introduced" include not only rice plants that have two alleles of the short anther gene with identical mutations, but also rice plants that have a first short anther gene with the first mutation encoding a protein with suppressed activity, and a second short anther gene with the second mutation encoding a protein with suppressed activity.

[0045] In the present invention, in addition to the above-mentioned introduction of mutations, other methods for artificially suppressing the function of the short anther gene include methods that target the transcript of the short anther gene, such as using DNA encoding dsRNA (double-stranded RNA, e.g., siRNA) complementary to the transcript of the short anther gene, using DNA encoding antisense RNA complementary to the transcript of the short anther gene (antisense DNA), and using DNA encoding RNA having ribozyme activity that specifically cleaves the transcript of the short anther gene (ribozyme method).

[0046] In the present invention, artificial suppression of the function of short anther genes can be performed on rice plants, seeds, or plant cells according to the methods described above. Plant cells include cultured cells derived from rice, as well as cells within the plant. Furthermore, various forms of rice-derived cells are included, such as suspension culture cells, protoplasts, leaf sections, callus, immature embryos, pollen, etc.

[0047] Furthermore, in the present invention, the above-mentioned site-specific nucleases, fusion proteins or DNA encoding a complex of guide RNA and protein, DNA encoding transposons, DNA encoding double-stranded RNA, DNA encoding antisense RNA, DNA encoding ribozyme-active RNA, etc., may be introduced into rice cells in the form of an inserted vector.

[0048] The vector into which the DNA for artificially suppressing the function of a short anther gene is inserted is not particularly limited as long as it is capable of expressing the inserted gene in rice cells, but it may contain a promoter for constitutive or inductive expression of the DNA. Examples of promoters for constitutive expression include the rice ubiquitin promoter, the cauliflower mosaic virus 35S promoter, the rice actin promoter, and the maize ubiquitin promoter. Examples of promoters for inductive expression include promoters known to be expressed by external factors such as infection or invasion by filamentous fungi, bacteria, or viruses, low temperature, high temperature, drought, ultraviolet irradiation, and spraying of specific compounds. Furthermore, as a promoter for expressing DNA encoding short RNA such as guide RNA or siRNA as the DNA of the present invention, polIII-type promoters such as the U6 promoter are preferably used.

[0049] Various methods known to those skilled in the art can be used to introduce the aforementioned DNA or a vector into rice cells, such as particle bombardment, Agrobacterium-mediated methods (Agrobacterium method), polyethylene glycol method, and electroporation.

[0050] Furthermore, even without taking the form of DNA, mutations can be introduced into rice cells by introducing the aforementioned site-specific nucleases, fusion proteins, and transposons as proteins, and by introducing the aforementioned guide RNA, double-stranded RNA, antisense RNA, and RNA with ribozyme activity as RNA.

[0051] Thus, in the present invention, the short-anther trait can be conferred to rice by using substances that target the short-anther gene, such as the DNA, the vector into which the DNA is inserted, the protein, and the RNA. Accordingly, the present invention can also provide a drug for conferring the short-anther trait to rice, which contains as an active ingredient at least one substance that targets the short-anther gene, selected from the group consisting of the DNA, the vector into which the DNA is inserted, the protein, and the RNA.

[0052] Such a drug may be configured to contain two active ingredients in a single composition, or it may be configured to contain two active ingredients in separate compositions (a so-called kit). In addition, the drug of the present invention may contain other components such as buffer solutions, stabilizers, preservatives, and antiseptics in addition to the above-mentioned substances.

[0053] Furthermore, by regenerating rice plants from cells in which gene function has been artificially suppressed using the methods described above, it is possible to obtain rice plants with the short anther trait.

[0054] For example, in rice, several techniques for producing transgenic plants have already been established and are widely used in the field of the present invention, including a method of regenerating plants by introducing genes into protoplasts using polyethylene glycol (Datta, SKIn Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) pp66-74, 1995), a method of regenerating plants by introducing genes into protoplasts using electrical pulses (Toki et al. Plant Physiol. 100, 1503-1507, 1992), a method of regenerating plants by directly introducing genes into cells using the particle gun method (Christou et al. Bio / technology, 9:957-962, 1991), and a method of regenerating plants by introducing genes via Agrobacterium (Hiei et al. Plant J. 6:271-282, 1994). Furthermore, transformation and regeneration into plants can be performed using the methods described in Tabei et al. (ed., Yutaka Tabei, "Transformation Protocols [Plant Edition]", Kagaku Dojin Co., Ltd., published September 20, 2012).

[0055] (Rice with short anther characteristics) By the methods described above, it is possible to obtain rice having the short anther trait in which the function of the short anther gene of the present invention is artificially suppressed. Therefore, the present invention is Rice with a short anther trait, in which the function of at least one gene selected from the group consisting of (a) to (d) below is artificially suppressed. (a) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 2. (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence described in Sequence ID No. 2. (c) A gene encoding an amino acid sequence having 95% or more homology to the amino acid sequence described in Sequence ID No. 2. (d) A gene containing DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence encoding the amino acid sequence described in Sequence ID No. 2. To provide.

[0056] As described above, the short anther gene, the artificial suppression of its function, and the rice in which the short anther trait is conferred by such suppression are as described above. However, the rice having the short anther trait of the present invention is preferably rice excluding san-1, and more preferably rice excluding the rice variety T65 (Taichung 65) in which the function of the short anther gene of the present invention is artificially suppressed.

[0057] Furthermore, once a plant in which the function of the short anther gene has been artificially suppressed is obtained, it is possible to obtain offspring from this plant through sexual or asexual reproduction. Moreover, it is possible to obtain reproductive materials (e.g., seeds, cuttings, stems, callus, protoplasts, etc.) from this plant, its offspring, or clones, and mass-produce the plant based on these materials. Therefore, the present invention includes offspring and clones of rice possessing the short anther trait, as well as their reproductive materials. Examples of reproductive materials include seeds, stems, callus, and protoplasts.

[0058] <Method for determining whether or not the short anther trait is present> The present invention provides a method for determining whether or not a rice variety possesses the short anther trait, characterized by analyzing the nucleotide sequence of the short anther gene or its expression regulatory region in the rice variety being tested. More specifically, it is as follows:

[0059] A method for determining whether or not a rice plant possesses the short anther trait, characterized by analyzing the nucleotide sequence of the short anther gene or its expression regulatory region in the test rice plant. The short anther gene to be detected in the determination method of the present invention is as described above.

[0060] As shown in the examples described later, the insertion or deletion of nucleotides in the short anther gene shortens the anther length. Therefore, by analyzing the nucleotide sequence of the short anther gene region, it is possible to determine whether or not a plant possesses the short anther trait.

[0061] Furthermore, by analyzing the expression level of the short anther gene, as well as the nucleotide sequence of the transcriptional regulatory region (enhancer, promoter, silencer, insulator, etc.) that controls its expression level, it is possible to determine whether or not a plant possesses the short anther trait.

[0062] When analyzing the nucleotide sequence of the short anther gene or its regulatory region, an amplified product obtained by PCR of the short anther gene or its regulatory region of the present invention can be used. When performing the PCR, the primers used are not limited as long as they can specifically amplify the short anther gene or its regulatory region, and can be appropriately designed based on the sequence information of the short anther gene or its regulatory region.

[0063] Furthermore, the method for determining whether or not a rice plant possesses the short anther trait may include, for example, a step of comparing it with a "control nucleotide sequence." The "control nucleotide sequence" to be compared with the nucleotide sequence of the short anther gene or its expression regulatory region in the rice plant is, in the case of rice, for example, the nucleotide sequence of the gene encoding the amino acid sequence described in Sequence ID No. 2 or its expression regulatory region.

[0064] By comparing the nucleotide sequence of the short anther gene or its expression regulatory region in the determined test rice with the nucleotide sequence of the control, it is possible to determine whether or not the test rice possesses the short anther trait. For example, if there is a significant difference in the nucleotide sequence compared to the control nucleotide sequence (e.g., SEQ ID NO: 1) (especially if the appearance of a new stop codon or frameshift causes a significant change in the molecular weight or amino acid sequence of the encoded protein), the test rice is likely to possess the short anther trait.

[0065] Furthermore, in the determination method of the present invention, the preparation of DNA from the test rice can be carried out by conventional methods, such as the CTAB method. Not only mature plants, but also seeds and young plants can be used as the plant for DNA preparation. The nucleotide sequence can be determined by conventional methods, such as the dideoxy method or the Maxam-Gilbert method. Commercially available sequencing kits and sequencers can be used for nucleotide sequence determination.

[0066] In addition to the direct sequencing described above, the nucleotide sequence of the short anther gene or its regulatory region in the test rice can be indirectly analyzed by various methods to determine whether it differs from the control nucleotide sequence. Examples of such methods include PCR-SSCP (single-strand conformation polymorphism), RFLP and PCR-RFLP methods utilizing restriction fragment length polymorphism (RFLP), denaturant gradient gel electrophoresis (DGGE), allele-specific oligonucleotide (ASO) hybridization, and ribonuclease A mismatch cleavage.

[0067] Another method for determining whether or not a rice variety possesses the short anther trait is characterized by detecting the expression or amplification product of the short anther gene or the molecular weight of the expression product in the rice variety being tested. More specifically, it is as follows:

[0068] A method for determining whether or not rice has a short anther trait, characterized by detecting the expression or amplification product of the short anther gene or the molecular weight of the expression product in the rice plant being tested. The short anther genes to be detected in the determination method of the present invention are as described above.

[0069] As shown in the examples described later, the insertion or deletion of nucleotides in the short anther gene reduces the molecular weight of the expression product and shortens the anther length. Therefore, the presence or absence of the short anther trait can be determined by detecting the molecular weight of the amplified or expressed product of the short anther gene. Furthermore, the presence or absence of the short anther trait can be determined by detecting the expression of the short anther gene.

[0070] Here, "detection of short anther gene expression" includes both detection at the transcriptional level and detection at the translational level. Furthermore, "detection of expression" includes not only the detection of whether or not expression is present, but also the detection of the degree of expression.

[0071] The detection of short anther genes at the transcriptional level can be performed by conventional methods, such as RT-PCR or Northern blotting. The primers used when performing the PCR are not limited as long as they can specifically amplify the DNA to be detected in the present invention, and can be appropriately designed based on the sequence information of the short anther genes that has already been determined.

[0072] On the other hand, detection at the translational level can be carried out by conventional methods, such as Western blotting. The antibodies used in Western blotting may be polyclonal or monoclonal antibodies, and the methods for preparing these antibodies are well known to those skilled in the art.

[0073] Based on the results of gene expression detection, if the expression level of the short anther gene in the test rice is significantly lower than that of the wild type (e.g., Nipponbare) in rice (for example, if the short anther gene is not substantially expressed), or if the molecular weight of the amplified or expressed product of the short anther gene is significantly different from that of the wild type, then it is determined that the plant has a high probability of possessing the short anther trait.

[0074] <Method for breeding rice with short anther traits> The present invention provides a method for breeding rice having the short anther trait. This breeding method includes the steps of (a) crossing a plant having the short anther trait with any variety, (b) The process includes selecting plants from among the individuals obtained by crossbreeding in step (a) that are determined to have the short anther trait by the method described above.

[0075] "Rice plants possessing the short anther trait" refers to, for example, rice plants that possess the short anther trait due to the suppression of the function of the san-1 short anther gene mentioned above. "Any variety" to be crossed with this plant could be, for example, a rice variety in which the function of the short anther gene is not suppressed and therefore does not possess the short anther trait, but is not limited to this. Furthermore, from the viewpoint of further suppressing cross-pollination, rice plants with the aforementioned cleistogamous pollination ability are preferred as the arbitrary variety. By using the breeding method of the present invention, it becomes possible to appropriately select rice varieties possessing the short anther trait at the young plant stage, and it becomes possible to develop varieties possessing this trait in a short period of time. [Examples]

[0076] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples.

[0077] (Example 1) Search for candidate causative genes of rice short anther mutants The rice san-1 (short anther) mutant was obtained from a population of mutants induced by the chemical mutagenesis substance N-Methyl-N-nitrosourea (MNU) using the rice variety T65 (Taichung 65) as the parent variety. The san-1 mutant exhibits anther shortening, with an anther length approximately 30% shorter than that of the wild type, while showing no difference in glume length (Figure 1, Non-Patent Literature 1).

[0078] However, the causative gene for this short-anther trait had not been identified. Therefore, we first crossed san-1 with the rice variety Kasalath and performed map-based cloning using the F2 population. As a result, we predicted that the causative gene is located between markers RM18639 and RM6841 on chromosome 5 (Figure 2).

[0079] After further intensive investigation and narrowing down the candidate region, it was predicted that the candidate region would be located between the markers IRIC11 and RM18719. Twenty genes were found within this candidate region in the RAP-DB database (https: / / rapdb.dna.affrc.go.jp / index.html). By comparing the gene sequences, it was found that in san-1, an immature stop codon is generated by the substitution of adenine at the 236th guanine position from the translation start site of the gene Os05g0421300 (Figure 3). This gene encodes a protein of unknown function, possessing a nuclear localization signal and an ARM (Armadillo repeat) domain at the N-terminus and a TPR (tetratricopeptide repeat) domain at the C-terminus (Figure 4).

[0080] (Example 2) Shortening of anthers by genome editing targeting gene Os05g0421300 To determine whether the candidate genes identified above are the causative genes for rice short-anther mutants, genome editing targeting these genes was performed using the method described below, and the expression of the short-anther trait was verified.

[0081] <Construction of vectors for expressing guide RNA and SpCas9, etc.> The genome sequence of gene Os05g0421300 was obtained from the RAP-DB database (https: / / rapdb.dna.affrc.go.jp / index.html). From the sequences predicted to be the first exon, two locations were selected as guide RNA sequences (20 bp long) to be targeted for genome editing. These were named SAN-CR1 (position: 376-395, sequence 5'-CCATTGGTTGAACTCTTACG-3', SEQ ID NO: 3) and SAN-CR2 (position: 137-156, sequence 5'-TTCTCCCTATTAGTGGTCTT-3', SEQ ID NO: 4).

[0082] Then, vectors (genome editing expression vectors) for expressing each guide RNA and SpCas9 were prepared according to standard methods. These vectors are based on the pZNH2GTR-U6 vector (Figure 5), with the DNA encoding the guide RNA inserted between the OsU6-2 promoter and the scaffold sequence. Furthermore, the DNA encoding SpCas9 is inserted between the rice Ubi1b promoter and the rice Ubi1b terminator sequence. This genome editing expression vector also contains a cassette sequence for expressing a hygromycin resistance gene for the selection of transformants.

[0083] <Creation and analysis of genome-edited rice> (1) Transformation into rice callus The genome editing expression vector prepared as described above was introduced into callus derived from rice embryodiscs of the rice variety Nipponbare by Agrobacterium-mediated transformation, according to the method described by Oikawa et al., Plant Mol. Biol. 55, 687-700 (2004). The transformed calluses were then selected by culturing them in a medium containing hygromycin.

[0084] (2) Extraction of genomic DNA from leaves Transgenic rice plants selected and redifferentiated using hygromycin were transplanted into culture medium (Bonsol No. 1). Approximately two weeks later, the tips of the elongated leaves (about 5 mm) were collected, placed in 1.5 mL plastic tubes, and DNA was extracted and subjected to PCR.

[0085] (3) Amplification of DNA fragments at the SAN target mutation site by PCR. The primer sequences used to amplify the target mutation site in plants that underwent genome editing using guide RNA SAN-CR1 or SAN-CR2 are as follows: Primers for SAN-CR1 analysis SAN_CR1_seqF 5'-TCAACTTGGGGATATTTGAATG-3'(Sequence ID: 5) SAN_CR1_seqR 5'-ACTTCTCCATGGTCAGCAACT-3' (Sequence ID: 6) Primers for SAN-CR2 analysis SAN_CR2_seqF 5'-TCATTTGTCTCTCACGATGGA-3' (Sequence ID: 7) SAN_CR2_seqR 5'-TGTGCTGCATAATATGGGATG-3' (Sequence ID: 8).

[0086] Tks Gflex DNA Polymerase (TAKARA Corporation) was used as the PCR enzyme, and the reaction mixture for a total volume of 10 μL was prepared as follows and carried out in an 8-tube strip with a volume of 0.2 mL. 2×Gflex PCR Buffer 5μL 100 μM Forward Primer 0.2 μL 100 μM Reverse Primer 0.2 μL Tks Gflex DNA Polymerase (1.25U / μL) 0.2μL Pure water 3.4μL 1 μL of DNA extract solution.

[0087] The PCR machine used was a TaKaRa PCR Thermal Cycler Dice and was operated under the following conditions. 94°C for 1 minute, then 98°C for 10 seconds, 55°C for 15 seconds, and 68°C for 30 seconds, repeated for 40 cycles.

[0088] (4) Sequencing analysis of PCR-amplified fragments PCR products from the region containing SAN-CR1 (wild-type, fragment length 301 bp) and the region containing SAN-CR2 (wild-type, fragment length 321 bp) were developed by 1% agarose gel electrophoresis, and amplification was confirmed. The amplified PCR products were purified using ExoSAP-IT Express (ThermoFisher Scientific), and direct sequencing analysis was performed using the forward primers used for amplification.

[0089] (5) Observation of anther length Anther length was observed in SAN genome-edited plants in the genetically fixed T1 generation. Pre-flowering glumes were collected, with the anthers extending to the middle of the glume. The outer glume of the collected glume was removed with tweezers to expose the anther, and the anther length was observed using a stereomicroscope.

[0090] <Analysis results of genome-edited rice> As described above, genome editing expression vectors were introduced into rice callus, and transformants were selected using hygromycin. Plants redifferentiated from the callus were transplanted into pots filled with growing medium and grown in an isolated greenhouse. Genomic DNA was extracted from the elongated leaves, and PCR amplification and sequencing analysis were performed to determine whether genome editing had occurred in each guide RNA region of the gene Os05g0421300.

[0091] As a result, as shown in Table 2, genome editing occurred in 83-85% of the T0 generation seedlings that were analyzed.

[0092] [Table 2]

[0093] Next, the plants in which genome editing was detected were transplanted into pots and cultivated in an isolated greenhouse to obtain progeny seeds. T1 generations were cultivated for line #20-1 (using SAN-CR1 as guide RNA) and line #14-1 (using SAN-CR2), and sequence analysis was performed on each individual to select individuals showing genome editing as homozygotes. As a result, it was found that in genome-edited plant san-CR1 #20-1, a single thymine base was inserted between the 392nd and 393rd bases from the translation start site of the gene Os05g0421300 (Figure 6), and in san-CR2 #14-1, a single thymine base was inserted between the 153rd and 154th bases (Figure 7), resulting in a frameshift.

[0094] Next, genome-edited individuals san-CR1 #20-1 and san-CR2 #14-1, which are homozygous for a frameshift-inducing mutation in the gene Os05g0421300, as well as individuals without genome editing, were grown in an isolated greenhouse, and glumes were collected before flowering. The outer glumes were then removed under a stereomicroscope to expose the anthers, and the anther lengths were compared.

[0095] As a result, compared to individuals without genome editing (the "vector control" in Figure 8), both san-CR1 #20-1 and san-CR2 #14-1 individuals showed approximately 30% shortening of the anthers (Figure 8).

[0096] Therefore, it was revealed that the gene Os05g0421300 is the gene responsible for anther shortening in san-1 (the SAN gene), and by targeting this gene and suppressing its function, it became possible to confer the anther shortening trait to rice.

[0097] (Example 3) Shortening of angiomas by genome editing targeting gene Os05g0421300 In Example 2 described above, genome editing was performed targeting the sequence predicted to be the first exon of the gene Os05g0421300 (SAN gene). This time, genome editing was performed targeting the sequence predicted to be the second exon, which is further downstream, and the presence and degree of anther shortening were evaluated. Specifically, the guide RNA sequence targeted for genome editing was SAN-CR3 (position: 1651-1670, sequence 5'-CATGGGGGAATCCATTGCGA-3', SEQ ID NO: 9). The genome-edited rice was created and analyzed in the same manner as in Example 2, except that the following primer set was used to amplify the target mutation site in the plant in which genome editing was induced using SAN-CR3. The results obtained are shown in Figures 9 and 10. Primers for SAN-CR3 analysis SAN_CR3_seqF 5'-GATATGCTTGGTTTAGCGAAAGAG-3' (Sequence ID: 10) SAN_CR3_seqR 5'-GTTCTTCCATTCAGTTTCATCATCT-3' (Sequence ID: 11).

[0098] As described above, a guide RNA (SAN_CR3) targeting the C-terminal side of the protein encoded by the SAN gene was designed, and a genome-edited organism was created. As a result, in the obtained genome-edited organism san-CR3#5, a frameshift occurred due to the insertion of thymine between the 1667th and 1668th bases from the translation start site (Figure 9). Furthermore, when the anther length of this genome-edited organism was observed, anther shortening was also observed in san-CR3#5 compared to individuals without genome editing ("control" in Figure 10). Moreover, the degree of anther shortening was weaker than in san-CR2#14-1, in which a mutation occurred at the N-terminal side (Figures 10 and 11).

[0099] Therefore, although the degree of anther shortening is weaker than that of san-CR2, in which a mutation is introduced further upstream in the SAN gene, it was confirmed that the anther shortening trait can be conferred to rice even by changing or deleting only about 7% of the amino acid sequence in the C-terminal region. Furthermore, it became clear that the degree of shortening can be controlled depending on the size of the altered or deleted region at the C-terminus of the amino acid sequence encoded by the SAN gene. [Industrial applicability]

[0100] As described above, the present invention makes it possible to produce rice with short anther characteristics. Furthermore, because of these short anther characteristics, the anthers are difficult to extract from the glumes and pollen is less likely to be dispersed, cross-pollination with other varieties can be suppressed. Therefore, the present invention is useful in the agricultural field related to rice.

Claims

1. A method for producing rice having the short anther trait, A method comprising the step of artificially suppressing the function of at least one gene in rice selected from the group consisting of (a) and (c) below. (a) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No.

2. (c) Sequence ID: A gene encoding an amino acid sequence that has 90% or more identity with the amino acid sequence described in Sequence ID No.

2.

2. Rice with a short anther trait, in which the function of at least one gene selected from the groups (a) and (c) below is artificially suppressed. (a) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No.

2. (c) Sequence ID: A gene encoding an amino acid sequence that has 90% or more identity with the amino acid sequence described in Sequence ID No. 2.

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